High-Level Expression of TGF-β2 and the TGF-β2(414) Precursor in Chinese Hamster Ovary Cells
作者:
MadisenLinda,
LioubinMario N.,
MarquardtHans,
PurchioA. F.,
期刊:
Growth Factors
(Taylor Available online 1990)
卷期:
Volume 3,
issue 2
页码: 129-138
ISSN:0897-7194
年代: 1990
DOI:10.3109/08977199009108275
出版商: Taylor&Francis
关键词: TGF-β2;in vitroexpression;immunoblotting;protein sequencing
数据来源: Taylor
摘要:
AbstractChinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-β2 (TGF-β2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-β2 precursor and subsequent amplification with methotrexate. The TGF-β2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-β2 indicated that correct processing of mature TGF-β2 had occurred. In addition to mature TGF-β2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serum-and cell-free media from recombinant CHO cells metabolically labeled with [3Hglucosamine and [32Porthophosphate indicated that pro-TGF-β2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hydrolysates showed that the32P was incorporated into mannose-6-phosphate.
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